Maize growth response to different Bacillus strains isolated from a salt-marshland area under salinity stress

Maize (Zea mays) growth performance has been hindered due to the high soil salinity. Salinity is one of the most severe abiotic stresses that has led to growth imbalance and profitability of harvests in arid and semi-arid regions. Plants have taken advantage of salt-tolerant bacteria as plant growth-promoters to enhance growth and reduce the adverse effects of salinity through the regulation of some biochemical, physiological, and molecular features. Preferences for non-chemical, eco-friendly, and economical approaches have caused the inquiry of the Bacillus genus as a joint group of plant growth-promoting rhizobacteria known to alleviate salt-stress impacts. In the present study, halotolerant Bacillus strains were isolated from salt-marshland soil and characterized for their physiological, molecular, and biochemical properties. Twenty-four bacterial isolates collected from high saline fields of salt marshland were analyzed by MALDI-TOF MS proteome analysis, which confirmed the taxonomic affiliation with Bacillus cereus, Bacillus subtilis, Bacillus atrophaeus, and Bacillus thorngiensis. Applying the isolates on maize plants as bio-inoculant bacteria obviously increased the growth parameters (P < 0.01). Pot experiments showed that isolates 74 and 90 were the most prominent strains to minimize the harmful effects of salinity. Its effects are heightening the potassium/sodium ratio and K-Na selectivity in shoots and roots measured by flame atomic absorption photometry (AAS). Accordingly, Bacillus cereus isolate 74 showed a maximum increase in dry weights of the shoot (133.89%), root (237.08%), length of the shoot (125%), and root (119.44%) compared to the control condition. Our findings suggest that bacteria isolated from marshland may be an economical and simple means to increase plant growth and resistance to high salinity soil conditions.


Introduction
Soil salinization is a widespread threat to agriculture and rangeland productivity in Iran. Salt marshes are extremely biodiverse habitats with complex structures [27] which play pivotal roles in the eco-geomorphological evolution [51]. These areas provide critical ecosystems for various plants, animals, and microbes that are adapted to deal with full stress macro-and microenvironments [23]. Coevolution of plants and rhizobacteria under intensive stress may provide a new insight into discovering halophilic bacterial agents to cope with the adverse effects of salinity on plant growth parameters. It seems that some halophilic bacterial strains positively affect plant growth promotion by improving their salt removal efficiency [5].
The present study aimed to isolate and characterize bacterial isolates collected from soil samples taken from one of the desert salt marshes in Iran. The potential of the bacterial isolates was assessed to promote maize growth parameters under salinity stress.

Soil Samples
Soil samples were collected from a desert marshland located at the north eastern of Qom (longitude 51 o 14'13.92" E and latitude 34 o 54'7.20" N). Eight soil samples were collected from 10 to 20 cm of the soil surface and completely mixed together and finally; one soil sample was transferred to the laboratory. The soil sample was dried in the dark and at room temperature for at least two days before sieving. Following the drying process, the soil sample was sieved through a 2 mm sieve to remove pebbles and other inert material and then kept in a zip lock cover.

Bacterial Isolation
Eleven culture media, including Nutrient Agar (NA), Nutrient Agar plus MnSO4 (NA + MnSO4), LB, Moller Hinton Agar (MHA), Acidithiobacillus (APH) medium, Violet Red Bile Lactose (VRB) agar medium, GYM Streptomyces medium, DPM medium, Azospirillum medium, and Azotobacter medium, were used. All media were sterilized at 121 °C for 20 min and plates were incubated inside polyethylene bags at 4 °C. For isolation of bacteria, 1 g of soil sample was suspended in 2 ml of sterile physiological saline (0.9% w/v NaCl) and then vortexed for 1 min. After sedimentation of soil particles, 100 ul of the supernatant was used for serial dilutions in the range of 10 −1 -10 −7 . Each dilution level was prepared in triplicate. Ten microliters of each dilution level were applied on the surface of solidified media by glass spreaders and incubated in an inverted position at 30 °C in the absence of light for 1-3 days. All instruments were sterilized completely before use and all dilution procedures were performed inside a laminar flow clean bench to achieve strict asepsis. Colonies were isolated based on the morphological characteristics (color, shape (top and side view), and diameter) using sterile toothpicks after two days of incubation at 30 °C. Accordingly, the number of each isolate was stated in colony-forming units per 1 g of soil (CFU g −1 ). Individual colonies were transferred and streaked separately on the same fresh media. Each plate was re-streaked twice to ensure strains purity and also cultured on the same liquid media for cryo-stock preparation. Long-term storage was carried out on a liquid medium containing 25% v/v glycerol at −70 °C.

Gram stain, Oxidase, Catalase, and KOH testes
Gram staining of bacteria was examined after 48 h of incubation on MHA following the method of Bartholomew [6]. A non-staining KOH method [43] was performed to confirm the results of Gram staining. A catalase test was performed using 0.5 ml of 10% hydrogen peroxide solution and observed for the formation of gas bubbles. The oxidative activity of 95 isolates was studied with biochemical oxidase disks.

Assessment of cold, dryness, salinity, heat, and pH on the bacterial growth
The growth of isolates (expressed as CFU) was evaluated in dark and under cold, drought, salinity, heat, and alkalinity stresses. Muller Hinton media was considered a basal growth media for all experiments. For cold and heat stresses, isolates were cultured at 15 and 60 °C for 10 hours, respectively. Basal media containing 100 mM NaCl, 25% PEG6000, and pH = 10 were prepared for salinity, drought, and alkalinity stresses and incubated for 10 h at 30 °C, respectively.

MALDI-TOF MS profile acquisition
Isolates were sub-cultured twice on MHA at 30 °C for 24 h. Then approximately 100 μg of the bacterial colony were directly transferred to the MALDI target spot. Followed by drying at room temperature and overloading with 1uL of matrix solution (10 mg/ml a-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid), each measurement was performed in two replications. MS analysis was carried out on an Autoflex MALDI-TOF mass spectrometer using Flex Control 3.4 software (Bruker Daltonics, Germany). Soil isolates with a valid MALDI-TOF MS score of 2 were undoubtedly assigned to the genus/species level.

Bacterial identification
The bacterial classification was carried out using Bio-Typer 3.1 software (Bruker Daltonics, Germany). All identifications were reported with the following score values; unreliable identification was <1.7, 1.7-2.0 considered a possible genus identification and 2.0-2.3 expressed as a secure genus identification and probable species identification; and finally, >2.3 was regarded as highly probable species identification. Only the highest score value of all mass spectra belonging to individual cultures (biological and technical replicates) was recorded [40].

Assessment of bacterial isolates on the maize growth
Maize seeds (Zea mays. Var Kosha) were obtained from Seed and Plant Improvement Institute of Karaj (Karaj, Iran, http:// www. spii. ir/ homep age. aspx? site= Doura nPort al& tabid= 1& lang= faIR), soaked in distilled water for 24 h and left to germinate. For the first irrigation, the sterilized MHA was treated with two salinity levels (0 and 100 mM NaCl) and bacterial isolates (CFU 2 × 10 3 ) and used as irrigation water. Each treatment consisted of one bacterial isolate and one salinity level. After the first irrigation, a daily plant watering (5 ml/ per day) was carried out using sterilized pure water. A completely randomized block design with factorial treatments in three replications was employed for the experiment. After 20 days, plants were harvested, and shoot and root length (cm) and shoot and root fresh weight (mg) were successively recorded. For dry weight, samples were dried at 50 °C and measured after reaching a stable weight. Whole dry weight (mg), whole length (cm), and Shoot/Root were calculated. Sodium and potassium contents were extracted as described by Sakr et al., [32] and measured with an Atomic Absorption Spectrometer (AAS).

Statistical analysis
All statistical analyses were performed by R software (version 3.6.2). The significance of the experiment was tested by one-way analysis of variance (ANOVA) and means separation was performed using Fisher's protected Least Significant Difference (LSD) test at P < 0.01 by package Agricolae. Pearson correlation analysis was performed using SPSS Statistics for Windows, version 16.0 (SPSS Inc., Chicago, Ill., USA) to check the level of association between control/salinity stress and relative growth of soil isolates. Cluster analysis was done based on the Tomida report [46] using CLUSTER (version 3.0) software, and tree images were performed by Java Treeview (version 1.1.6r4). Hierarchical clustering was performed according to the Euclidian distance and complete linkage method.

Isolation, characterization, and MALDI-TOF MS-based identification
Of 24 isolates, seven isolates were obtained on MHA, 2 on NA, 3 on NA + , 3 on LB, 2 on VRB, 3 on AZTO, and 4 on GYM through the morphological distinctions. All isolates presented a rode shape in cell form with mostly smooth and flat surface colony shape except for two isolates (35 and 120) by screening them on selective media. The isolates came in a variety of colors, including cream, white, yellow, orange, brick red, and red. Table 1 indicates soil isolates growth rates under selective media (e.i., MHA, NA, NA + , LB, VRB, AZTO, and GYM). Table 2 shows the effect of abiotic stresses on bacterial growth. Accordingly, under salinity stress, isolates 36, 105, 106, 120, 121, and 128 showed growth equal to or greater than normal conditions. Although isolates 33, 35, 39, 73, 74, 90, 129, 130, and 149 also had significant growth under salinity stress, their growth retardation was minimal under salinity stress. In cold stress, only isolate 36 could grow higher than the control, and the rest of the isolates had reduced growth. Under heat stress, the growth of isolate 146 was 1.3 times higher than the control under stress conditions, but the rest of the samples had a considerable growth reduction. Under heat stress, the development of 146 isolates was 1.3 times higher than the control under stress conditions, but the rest of the samples had a large growth reduction. At a higher pH level (pH = 10), isolates 89 and 146 showed better growth under stress than normal conditions, and the development of the rest isolates was decreased; however, isolates 90, 147, and 148 had acceptable change compared to controls. Also, all isolates under drought stress had severe growth retardation.
Molecular characterization and biochemical analysis were done on bacterial isolates with catalase, KOH degradation, and oxidase abilities. All 24 isolates were shown Gram-positive and minus catalase reactions. However, there were differences between the isolates for oxidase and KOH degradation tests. In this regard, only four isolates showed KOH digestion activity (33,36,104, and 130) and six isolates did not show oxidase activity (37, 90, 121, 128, 130, and 147). Table 3 summarizes the results of the identification of isolates by the MALDI-TOF method and the results revealed a close relatedness of the isolates to the Bacillus genus. Protem analysis of the isolates produced an exact match to four species during MALDI-TOF and the results showed all the isolates belonged to B.cereus, B atrophaeus, B.subtilis, and B.thuringiensis. Besides, the results of biochemical tests and Gram staining confirmed the results of MALDI-TOF. In this way, all isolates were circular, Gram-positive, and produced endospores; so, they were also characterized based on distinguishing features.

Effect of isolated bacterial strain on Maize plant growth parameters under control conditions and salinity stress
The results of the application of isolates on plants indicated that yield per plant and growth parameters were significantly boosted by bacterial inoculation under salinity stress conditions; so isolates could alleviate the deleterious impacts of salinity on the growth of maize plants under natural salinity field conditions. Shoot dry weight, root dry weight, shoot length, and root length readings in maize leaves and roots were significantly increased by bacterial isolates under salinity stress. Hence, bacterial assessments improved the growth parameters of maize plants compared with the non-inoculated control also under salinity stress.
The results of tests under salinity conditions showed distinct differences in the shoot and root growth between isolates' treatments. Plant-microbe interactions under salinity stress were statistically significant (p < 0.001). Four growth parameters were associated with salt tolerance analysis in maize and the analyses were linked to shoot dry weight, root dry weight, shoot length, and root length of maize plants under normal and salinity conditions. Table 4 demonstrated the effect of soil isolates on maize plants' growth under normal conditions and salinity stress. As the results showed, isolates 33, 74, 90, and 130 obviously have boosted dry weight under stress conditions compared to the control, whereby a strong increase in root length and weight were recorded. Also, isolates 33, 74, 90, 105, 130, and 128 showed better effects Isolates 74 and 130 possessed a higher shoot length under stress than control conditions (Fig. 1a,b) and isolates of 33, 74, 130, and 146 had the same impact on the length of root in maize plants. Moreover, isolates 90 and 130 also showed the highest effect on plant length (shoot + roots) under stress conditions compared to the control. Isolate 105 also was more effective in shoot length, and this could be due to the induction of auxin production by Bacillus isolate 105.

The Na + and K + levels in shoot and root in both salinity and normal conditions
The highest values for total plant biomass were found in the plants inoculated with isolates 74 and 90 under both non-saline conditions and NaCl stress (Table 5). According to the results, the Na + and K + contents in the shoots and roots were affected by bacterial application under both saline and non-saline conditions, with a significant reduction in Na + levels in the leaves and roots compared to the un-inoculated control. At 100 mM NaCl, theresults of LSD analysis of Na and K contents in shoots and roots of maize plants were shown a significant difference between control and salt stress conditions at the level of P < 0.001. Table 5 represents the amounts of sodium and potassium ions in the root and leaf tissue of maize under normal and salinity stress. As shown, the amount of Na was at the lowest level in maize plants at 100 mmol/l NaCl salinity condition, which was inoculated with isolate 74; so isolate 74 led to less salt absorbance. Also, the foliar and fibroid levels of K increased with the inoculation of this bacterial isolate.

Pearson correlation
Research efforts investigating the effects of salt-marsh isolates on plants grown under salinity stress have led to the characterization of different microbial behaviors under stress conditions, which involve their roles in creating salinity tolerance in maize plants. The Pearson correlation coefficient between plant growth parameters under control and salinity stress and bacterial growth in the control (Table 6) and stresses (Table 7) conditions reflects a significant positive correlation between shoot dry weight and other plant growth parameters under control and salinity stress (Tables 6 and 7). Furthermore, bacteria growth under salt stress had a great correlation with its growth under drought and cold stress (Tables 6  and 7) as well bacterial growth under heat and pH stresses (Tables 6 and 7) under both salinity and control conditions.

Cluster analysis of plant responses to salinity under inoculation of 24 soil isolates
The abiotic stress caused by enhancing chemicals such as Na is a common phenomenon occurring in the rhizosphere. It is responsible for the presence of a wide variety of physiological processes in plants. The application of the salt-tolerant bacteria, which naturally reside in the rhizosphere as plant growth promoters, provides a natural way to boost plant species' growth and resistance. The results of clustering of plant growth under salinity stress in the presence and absence of isolates showed that, in terms of affinity, the growth parameters WW, RDW, and RL come together in one group, and ShW, ShL, and WL in another group (Fig. 2). Isolates are also classified into two main groups in terms of their effect on plant growth. The first group showed better effectiveness on ShL, WL, and ShW, and the second group was more effective on RW, WW, and RL (Fig. 2).

Discussion
Several abiotic elements may play a key role in modulating the diversity of soil microbes, including those inhabiting the rhizosphere [34], and one of the most important abiotic stresses is high-salt concentration. According to the previous reports, approximately 831 million hectares of the land area are affected by salt worldwide [39]. Salinity impedes photosynthesis and increases photorespiration, thereby altering the normal ion homeostasis of cells by providing nutrient imbalance, which is caused by loss of the plant's ability to control nutrient uptake and/or transport from root to shoot, leading to ion deficiencies [35]. Microbial forms of life can be found over a vast range of salt concentrations. Furthermore, these organisms cope with hyperosmotic stress by utilizing various strategies [41] to mitigate salinity and boost the efficiency of plants [9]. Among bacteria, the genera bacilli and pseudomonas are two of the most critical and common plant growth promoters [9,19,21,24,30]. Also, it has been demonstrated that the existence of indigenous marshland isolates is still important to study [42], as it will open an opportunity to develop local-strain-based plant promoters' productions against abiotic stresses such as salinity which is very common in Iran. Several reports have indicated that the sequences of dominant Bacillus species present in different soils are not the same as those present in easily cultured isolates [55]. It was demonstrated that not only Bacilli isolates could enhance the accumulation of amino acids and carbohydrates but also reduce the antioxidant activity of     enzymes such as catalase and glutathione peroxidase and electrolyte leakage in Zea mays [48]. Root-associated microorganisms play a critical role in retaining soil humidity and promoting plant growth under abiotic stresses by available services like microbial ecological services, protection from the soil mechanical stress, protection from osmotic and oxidative stresses, and effects on hormone homeostasis [11].
The findings of the present study revealed that shoot, root dry weight per plant, and length of shoot and root were significantly increased by bacterial applications under salinity stress conditions. Inoculation of isolates significantly (P < 0.01) increased shoot dry weight and root dry weight, which are useful measures of the physiological stability of plants. The exploitation of soil microbes for utilizing salt-stressed land is a useful method that may provide a quick-fix solution to salinity [44] as marshland microbes show different types of metabolic and adaptive responses to the variable supply of water, oxygen, organic/inorganic substrates and other available nutrients [4,15]. The effect of isolates on the growth parameters of maize under salinity stress proved that bacteria were divided into three groups in terms of effectiveness: a) Isolates with a favorable effect on plant growth, b) Isolates with adverse effects on plant growth, and c) Isolates with no impact on plant growth. Hence it has been proved that bacteria of the Bacillus genus have been widely reported as promising candidates for bacterization because of their ability to eliminate or alleviate the harmful effects of saline stress, regulate plant physiological characteristics, and promote plant growth [14,19,29,49,53,56]. Also, Wang et al. [50] reported increased shoot length of capsicum at 300 mM salt concentration when treated with B. megaterium.
In summary, the findings of the current study suggest that bacterial isolates belonging to Bacillus are dominant in the rhizosphere of salt marshland. Under salinity stress, 29% of isolates had a significant effect on shoot weight, 50% on root weight, 16% on shoot length, and 8% on root length. It has been established Fig. 2 Represents Hierarchical cluster analysis of the effect of isolates on maize plants under control and salinity stress (100 mM salt stress) drown by CLUSTER and Treeview softwares. Hierarchical clustering was done based on the Euclidian distance and complete linkage method. Colors indicate the impacts of isolates on the plants. Accordingly, red, green, and black colors show positive, negative, and no-effect isolates, respectively. The horizontal axis indicate plant growth parameters: Whole weight (WW), Root dry weight (RDW), Root length (RL), Shoot dry weight (ShDW), Shoot length (ShL), Whole length (WL). Vertical axis showes the assayed bacterial isolates those high concentrations of sodium and chloride in soil may depress nutrient ion activities and produce extreme ratios of Na/Ca and Na/K in the plants causing the plants to be susceptible to osmotic and specific ion injury, as well as to nutritional disorders, resulting in reduced yield and quality [16,45]. Based on our findings, the observed increase in the maize growth variety on inoculation with Bacillus sp under salt stress has been supported by reducing the Na/K ratio. Hence our results showed isolates 74, 33, and 91 reduced this ratio and increased salt tolerance in maize plants. Potassium (K) plays a crucial role in water stress tolerance in salinity stress. It is an osmotically active compound that contributes to water absorption in the root [17,20]. AbdElgawad et al. reported that maize plants could increase enzymatic and nonenzymatic antioxidants under salt stress to prevent harmful effects on salinity [1]. Also, it was shown that increase in phosphatidylglycerol, as well as a decrease in phosphatidylethanolamine and linoleic acid, correlated with salt tolerance in Zea mays [33]. Accordingly, we conclude that bacterial isolates are involved in salinity tolerance in a way to aids the plant in boosting such antioxidants and lipids.
Indeed, salt marsh sediment bacteria remain primarily in a black box regarding their diversity and functional roles through salt marsh benthic food web pathways [12]. Therefore, we investigated whether it is possible to isolate aerobic bacteria with efficacy for salt tolerance from salt-marshland and whether these bacteria can also induce salinity tolerance in maize plants. We conducted this study on a salt marsh because the spatial ecology of salt marshes is exceptionally well understood; so bacteria exhibited in salt marshes are important in many ways. For instance, bacterial isolates from the rhizosphere of salt marsh show host specificities on plant vegetation in terms of composition [7] as well as in terms of abundance and heterotrophic activity [7,10]; thus reflecting the adaptation to distinct environmental pressures. These shreds of evidence were confirmed by the data in the present study showing that salt-marshes are profitable and dynamic ecosystems with chemical and physical gradients that lead to discovering aerobic bacteria with salt tolerance efficacy, which can mitigate deleterious effects of salt stress and also induce salinity tolerance in plants.

Conclusion
Assessing Z. mays plants with beneficial soil bacterial isolates have a great impact on plant growth and their persistence over abiotic stresses such as salinity. It is important to find practical approaches to isolate and characterize such bacteria. One of the most critical steps is how to figure out the correct place to isolate these bacteria. The particular conditions of salt marshlands can provide a unique environment for the emergence of salt-resistant bacteria. Many of these kinds of isolates, as well as resisting salt stress, may create resistance in surrounding organisms. The main challenge in this study was to survey the effectiveness of isolates on plant growth and their salt resistance. Our findings indicate that B. subtilis 73, B. cereus 74, and B. atrophaeus 90 are talented salt-marshland strains that need to be more analyzed in further studies for their application feasibility on the maize crop.